Estradiol Receptors Inhibit Long-Term Potentiation in the Dorsomedial Striatum

Abstract Estradiol, a female sex hormone and the predominant form of estrogen, has diverse effects throughout the brain including in learning and memory. Estradiol modulates several types of learning that depend on the dorsomedial striatum (DMS), a subregion of the basal ganglia involved in goal-directed learning, cued action-selection, and motor skills. A cellular basis of learning is synaptic plasticity, and the presence of extranuclear estradiol receptors ERα, ERβ, and G-protein-coupled estrogen receptor (GPER) throughout the DMS suggests that estradiol may influence rapid cellular actions including those involved in plasticity. To test whether estradiol affects synaptic plasticity in the DMS, corticostriatal long-term potentiation (LTP) was induced using theta-burst stimulation (TBS) in ex vivo brain slices from intact male and female C57BL/6 mice. Extracellular field recordings showed that female mice in the diestrous stage of the estrous cycle exhibited LTP similar to male mice, while female mice in estrus did not exhibit LTP. Furthermore, antagonists of ERα or GPER rescued LTP in estrous females and agonists of ERα or GPER reduced LTP in diestrous females. In males, activating ERα but not GPER reduced LTP. These results uncover an inhibitory action of estradiol receptors on cellular learning in the DMS and suggest a cellular mechanism underlying the impairment in certain types of DMS-based learning observed in the presence of high estradiol. Because of the dorsal striatum’s role in substance use disorders, these findings may provide a mechanism underlying an estradiol-mediated progression from goal-directed to habitual drug use.


Introduction
Estradiol, the primary form of the sex hormone class estrogen, has a number of neurologic functions, including a role in modulating learning that is associated with the dorsal striatum. This brain region is part of the basal ganglia and is involved in motor skill learning, goal-directed behavior, habit learning, and drug abuse. While sensorimotor performance is enhanced in the presence of estradiol (Becker et al., 1987), several types of learning that involve dorsal striatal circuits are impaired, such as cued learning and response learning (Zurkovsky et al., 2007;V.C. Wang et al., 2009;Neese et al., 2010).
Estradiol is also known to influence the course of addictive behavior, which heavily involves dorsal striatal circuits. Dorsal striatal activity is involved in acquisition of drug-seeking and drug-taking behaviors. During the development of substance use disorders, control over drug-taking shifts from goal-directed learning in the dorsomedial striatum (DMS) to habit-based learning in the dorsolateral striatum (DLS) as drug use switches from recreational to compulsive (Yin et al., 2009;Corbit et al., 2012;Murray et al., 2012;Gremel and Costa, 2013;Lipton et al., 2019). Higher estradiol leads to more rapid drug-taking behavior in female rats, and female rats acquire self-administration of drugs more rapidly than male rats (Lynch and Carroll, 1999;Jackson et al., 2006;Becker and Hu, 2008). In humans, observations among individuals susceptible to substance use disorders reveal that women progress into habitual drug use more quickly than men (Piazza et al., 1989;Becker, 2016;. The more rapid development of habitual drug-taking may be because of weaker control of reward-sensitive, goal-directed behavior by the DMS. Understanding the effects of estradiol in the DMS will further the understanding of gender differences in substance use disorder. The three main types of estradiol receptors, ERa, ERb , and G-protein-coupled estrogen receptor (GPER), are found throughout the dorsal striatum, but their mechanism of action in pathways underlying learning in this area is not well understood. The receptors are found exclusively outside the nucleus in dorsal striatal neurons of adult mice (Almey et al., 2012), suggesting nongenomic effects. Studies on other brain regions have found that estradiol receptors acting through intracellular signaling pathways can affect spine growth, receptor trafficking, and protein synthesis (Taxier et al., 2020). However, studies of the effect of estradiol on dorsal striatal processes are limited. ERa and ERb are clustered with mGluR3 (G i/o -coupled receptor), and ERa also is clustered separately with mGluR5 (G q -coupled receptor; Grove-Strawser et al., 2010). The pathways activated by ER interactions with mGluR3 and mGluR5 have opposing actions on L-type calcium currents (Mermelstein et al., 1996) and cAMP response element-binding protein (CREB) phosphorylation (Grove-Strawser et al., 2010). The third ER type, GPER, leads to activation of the Gs-coupled signaling pathway (Thomas et al., 2010;Evans et al., 2016;Yu et al., 2018). It is unknown what role, if any, these ERs play in corticostriatal plasticity. While one study has found that estradiol receptor activity is necessary for long-term potentiation (LTP) in the DLS of male mice (Tozzi et al., 2015), it is unknown whether this is also the case in the DMS or in female mice. The aforementioned findings of impairment by estradiol on dorsal striatal-based learning in females suggest that the hormone may have an inhibitory effect on corticostriatal LTP.
This study tested the hypothesis that estradiol during estrus impairs plasticity in the DMS of female mice. A theta-burst stimulation (TBS) protocol was used to induce corticostriatal LTP in the DMS of C57BL/6 male and female mice. Results demonstrate that male mice and female mice in the low estradiol stage of diestrus undergo LTP, but female mice in estrus do not. Estradiol receptor agonists and antagonists were then used to determine the role of estradiol receptors in this LTP effect. It was found that blocking either ERa or GPER rescued LTP in estrous mice and activating either ERa or GPER inhibited LTP in diestrous mice. In male mice, on the other hand, ERa activation blocked LTP but GPER activation did not. The findings indicate that estradiol impairs DMS synaptic plasticity through the actions of ERa in males, and through both ERa and GPER in females.

Materials and Methods
All animal procedures were performed in accordance with George Mason University's institutional animal care and use committee (IACUC) regulation and followed the National Institutes of Health animal welfare standards. Every measure was taken to minimize stress and the number of mice used. C57BL/6 mice were group-housed in Teklad 7099 TEK-Fresh (wood pulp) bedding with samesex littermates in a standard 12/12 h light/dark cycle in a light-controlled and temperature-controlled facility with ad libitum access to food and water.
Estrous status in female mice was determined through cytological assessment of vaginal smears collected through lavage immediately following decapitation, which occurred between the hours of 12 and 2 P.M. Diestrus was characterized by a high prevalence of leukocytes and an absence or low prevalence of cornified or nucleated epithelial cells. Estrus was characterized by a high prevalence of cornified epithelial cells, an absence or low prevalence of nucleated epithelial cells, and an absence of leukocytes.
Slices were perfused with 30-32°C oxygenated ACSF containing 50 mM picrotoxin (Tocris Bioscience) at a rate of 2.5 ml/min in a submersion recording chamber. A tungsten bipolar stimulating electrode (A-M Systems, 796500) was used to inject current into the corpus callosum overlying the dorsomedial striatum (DMS; Fig. 1A). Current was injected in pulses of 0.05 ms. Field recordings of the voltage response from a population of neurons (pop spike) within the DMS were recorded using a borosilicate glass pipette (Sutter Instrument, BF150-75-10) containing ACSF.
Pop spike amplitude was measured by taking the absolute value of the difference between the negative peak of the trace after the stimulation artifact and either the positive peak immediately preceding the negative peak or the mean voltage of the trace immediately before the stimulation artifact, whichever was greater (Lovinger et al., 1993;Hawes et al., 2013). The signal was filtered through a fourpole, low pass Bessel filter with a cutoff frequency of 5 kHz (Warner LPF-100B), amplified by an electrometer (Warner IE-251A), and sampled at 20 kHz using LabView.
In order to determine the current amplitude to use for each experiment, an input-output curve was conducted, measuring the pop spike response across a range of current amplitudes (Fig. 1B). The current that would produce 40-60% of maximal response was selected for the remainder of the experiment.
The recording protocol using this selected current amplitude was comprised of three sections: a baseline recording, theta-burst stimulation, and a follow-up recording (Fig. 1C). The baseline pop spike recording was performed by taking measurements of pop spikes in response to a pulse every 30 s. This was run until the baseline had no discernable slope for at least 15 min. Experiments in which the baseline slope during the last 15 min exceeded 60.01/min were excluded from analysis. Experiments with average baseline sizes exceeding 1.3 mV were also excluded.
The baseline was followed by TBS to induce long-term potentiation (LTP; Hawes et al., 2013;Fig. 1D). The TBS protocol consisted of 10 trains of 10 bursts with four pulses in each burst. The inter-train interval was 15 s. The interburst interval was set at a frequency of 10.5 Hz and the interpulse interval at 50 Hz. Finally, a follow-up was conducted for 30 min using the same, 0.033-Hz pulse frequency as the baseline.

Results
Long-term potentiation does not occur in the DMS of female mice in estrus Dorsal striatal-based behaviors and learning differ between males and females and, as seen in rodents, across stages of the estrous cycle where estradiol fluctuates (Korol and Pisani, 2015;Becker, 2016;Meitzen et al., 2018). Since LTP is a cellular mechanism of learning, this study investigated whether the observed behavioral differences may be associated with differences in LTP between sexes or across the estrous cycle. LTP was induced using a TBS protocol in ex vivo coronal brain slices of male, female diestrous, and female estrous mice. Pop spikes before and after LTP induction were measured using extracellular field recordings. Estrous status was determined using vaginal cytology following decapitation. Uteri were extracted and weighed 15-20 min after decapitation. A Wilcoxon two-sample test determined that the mice that were categorized into the estrus stage had significantly higher uterus weights than those categorized into diestrus, Z ¼ À4.38, p , 0.0001 a (Fig. 2), confirming the assessment of estrous status.
Before collecting a baseline pop spike amplitude, an input-output curve was measured to select the current that produced 40-60% of maximal response. The shape of the input-output curve appeared different across sexes and estrous status: males had the greatest pop spike response to higher currents and estrous females had the lowest (Fig. 1B). Consequently, the mean baseline pop spike size differed among these groups: a one-factor generalized linear model showed a significant difference among males, diestrous females, and estrous females, F (3,106) ¼ 3.29, p ¼ 0.041 b . A Tukey-Kramer post hoc test revealed the estrous group had a lower mean baseline pop spike amplitude than males (p ¼ 0.032 b1 ) but not diestrous females (p ¼ 0.23 b2 ). Male and diestrous female baselines did not differ (p ¼ 0.70 b3 ). To ensure that the baseline size itself was not producing differences among groups, for each section below, baseline size was compared among groups, and for those with differences, the effect of baseline pop spike amplitude on LTP was tested. Males and diestrous females both exhibited LTP, where normalized pop spike size increased ;25% from baseline in males and ;40% in diestrus females, but LTP was not observed in estrous females ( Fig. 3A-C). The estrous groups exhibited a short-term depression lasting ;10 min after induction. A one-factor generalized linear model across male, diestrous female, and estrous female groups revealed an overall significant difference between groups at 30 min after induction (F (2,24) ¼ 6.29, p ¼ 0.0064 c ). Tukey-Kramer post hoc comparisons indicated that at 30 min, normalized pop spike size was significantly greater in diestrous females (p ¼ 0.0062 c1 ) than in estrous females (Fig. 3C). Pop spike size at 30 min did not differ between males and diestrous females (p ¼ 0.34 c2 ) or males and estrous females (p ¼ 0.053 c3 ). This indicated that LTP occurred following TBS in diestrous females but not in estrous females.
To verify that estrus was the factor impairing LTP, additional tests assessed whether baseline pop spike amplitude differed between groups and whether mouse age influenced pop spike size at 30 min. A one-way generalized linear model showed no difference in baseline pop spike size between male, diestrous female, and estrous female groups in the no drug condition (F (2,24) ¼ 0.97, p ¼ 0.39 d ). An analysis of covariance was run using sex and estrous status as factor, and age as covariate to determine whether age predicted pop spike amplitude at 30 min. This test showed that age was not significant, (F (1,23) ¼ 0.09, p ¼ 0.77 e ). These results demonstrate that estrous status, and not baseline size or age, is responsible for group differences in LTP.

ERa and GPER inhibit corticostriatal long-term potentiation in female mice
Estradiol in the striatum and other brain tissue correlates with blood plasma levels (Bixo et al., 1986); thus, it was expected that estradiol receptor (ER) activation was lower in brain slice during diestrus, a stage when estradiol levels are lowest, than during estrus. Accordingly, based on the above finding that LTP in the DMS could be induced during diestrus but not during estrus, it was hypothesized that this hormone's actions on ERs have inhibitory effects on LTP. To test this hypothesis, TBS was run in the presence of ER antagonists and agonists to determine the ER subtypes responsible for inhibiting LTP.  First, this study tested whether LTP could be rescued in estrous females using antagonists of each of ERa, ERb , or G-protein-coupled ER (GPER). The results were compared with the no drug TBS condition (Fig. 4A,B). A significant difference was found using a one-factor generalized linear model across estrous groups at 30 min (F (3,26) ¼ 6.15, p ¼ 0.0026 f ). A post hoc test using Dunnett adjustment for multiple comparisons revealed that the normalized pop spike at 30 min was significantly higher with the ERa antagonist MPP (p ¼ 0.044 f1 ) and the GPER antagonist G15 (p ¼ 0.0085 f2 ) than the no drug control, while the ERb antagonist PHTPP had no effect (p ¼ 0.94 f3 ). The G15 antagonist also prevented the short-term depression seen in the no drug condition. Baseline pop spike amplitude did not differ among these estrus groups (F (3,26) ¼ 1.71, p ¼ 0.19 g ). Thus, TBS-induced corticostriatal LTP can only be induced in the absence of either ERa or GPER activity.
In diestrous females, TBS was run in the presence of ERa or GPER agonists to mimic a higher estradiol level and determine whether LTP would be inhibited by actions of a single ER type. Induction of LTP at 30 min was compared with the no drug diestrous group (Fig. 5A,B). ERb agonism was excluded because of nonsignificant findings from the antagonist (Fig. 4). A main effect of drug was found using a one-factor generalized linear model (F (3,29) ¼ 5.27, p ¼ 0.0050 h ). A post hoc test using Dunnett adjustment for multiple comparisons determined that the ERa agonist PPT (p ¼ 0.0052 h1 ) and the GPER agonist G1 (p ¼ 0.017 h2 ) had a significantly lower pop spike magnitude than the no drug control. Because neither agonist completely blocked LTP, additional recordings were performed in the presence of the two agonists combined. LTP was further reduced in the presence of both PPT and GPER (p ¼ 0.0026 h3 ). Baseline pop spike amplitude did not differ among these diestrous groups (F (3,29) ¼ 0.84, p ¼ 0.48 i ). Therefore, both ERa and GPER activity inhibits LTP in the dorsomedial striatum of female mice.

ERa inhibits corticostriatal long-term potentiation in male mice
The next question was whether the same effects of ERa and GPER activation and antagonism on corticostriatal LTP would be seen in male mice (Fig. 6). Males were tested because of the presence of circulating estradiol, though their levels are lower than observed in intact females (Morissette and Di Paolo, 1993;Fester et al., 2012). Additionally, males have aromatase in the dorsal striatum, indicating that there is local production of estradiol there (Küppers and Beyer, 1998). They also express similar levels of ERs as females (Küppers and Beyer, 1999;. Both agonists and antagonists were tested because, unlike in females, estradiol does not fluctuate in males, thus an increase or decrease in ER activity may shift male corticostriatal plasticity. First, the role of ERa in LTP in males was tested. The ERa agonist PPT resulted in a clear decrease in pop spike size from the no drug condition (Fig. 6A), similar to what was observed in diestrous females (Fig. 5). The ERa antagonist MPP slightly reduced pop spike size (Fig. 4). To determine whether these observed differences were significant, a one-factor generalized linear model was run and a main effect of ERa drug was found (F (2,25) ¼ 4.06, p ¼ 0.030 j ). Dunnett's post hoc comparisons indicated that the ERa agonist PPT produced a significantly lower pop spike size at 30 min than the no drug control (p ¼ 0.017 j1 ). The antagonist MPP did not significantly change pop spike size from the no drug control group (p ¼ 0.38 j2 ; Fig. 6C). Baseline pop spike size differed among the male groups (F (2,25) ¼ 9.21, p ¼ 0.001 k ), where the no drug condition was significantly lower than the PPT (p ¼ 0.0041 k1 ) and MPP (p ¼ 0.0039 k2 ) groups. Despite this difference, pop spike size at 30 min was not predicted by baseline pop spike amplitude (F (1,24) ¼ 0.16, p ¼ 0.70 l ). This indicates that ERa activation inhibits LTP in the male mouse DMS but, as expected in low levels of estradiol, LTP is not enhanced on inhibition of ERa.
When LTP induction was tested in the presence of the GPER agonist G1 or the GPER antagonist G15, both drugs appeared to decrease pop spike size by about half (Fig. 6D). However, statistical analysis using a one-factor generalized linear model revealed no differences between the no drug condition and the G1 and G15 groups (F (2,27) ¼ 2.50, p ¼ 0.10 m ; Fig. 6F). These results indicate that, unlike in females, GPER activation does not prevent LTP induction in male mice.

Discussion
This study examined the effect of the estrous cycle and estradiol receptors (ERs) on synaptic plasticity in the dorsomedial striatum (DMS). The results showed that longterm potentiation (LTP) can be induced using theta-burst stimulation (TBS) in the DMS of diestrous mice, and that the amplitude is comparable to male mice, but that LTP induction is hindered during the estrous stage of the cycle. Experiments further demonstrated that ERa and GPER are modulating LTP induction. Specifically, corticostriatal LTP occurs in estrous mice when either ERa or GPER is blocked. In diestrous mice, when circulating estradiol levels are low, using an ERa or GPER agonist impairs LTP. In male mice, the ERa agonist impairs LTP but the GPER agonist does not, and neither the ERa nor the GPER antagonist has a significant effect. Collectively, these findings demonstrate a novel role of estradiol in corticostriatal LTP and offer a new context for the current understanding of the hormone's actions in the DMS.
Estradiol may be controlling LTP through its modulation of striatal neuron properties. Spiny projection neurons (SPNs) in the dorsal striatum of mice exhibit reduced excitability during estrus compared with other stages of the cycle, as evidenced by a higher rheobase, higher action potential threshold, and lower action potential amplitude (Willett et al., 2020). The role that ERs play in these changes is unclear. In the rat ventral striatum, miniature EPSC (mEPSC) frequency fluctuates across the estrous cycle and estradiol modifies mEPSC frequency and neuron excitability (Proaño et al., 2018(Proaño et al., , 2020Krentzel et al., 2019). However, in the rat dorsal striatum, no effect of estradiol on excitability has been observed, regardless of estrous status (Krentzel et al., 2019). These observations in the rat striatum do not imply that estradiol does not play a role in mouse dorsal striatal SPN properties: there may be an interspecies difference in estradiol action on SPNs, or it is possible that evoked, instead of spontaneous, synaptic input is altered by estradiol. Alternatively, while SPNs are a logical target to study estradiol's effects in the striatum because they comprise over 90% of striatal neurons (Kemp and Powell, 1971;Bolam et al., 2000), it cannot be ruled out that the effect of estrus is because of estradiol action on interneurons, although a recent related study found no evidence of estradiol affecting cholinergic interneuron excitability (Kövesdi et al., 2023). Another possibility is that estradiol's effect on striatal circuitry underlying LTP may come from another source, such as dopamine neurotransmission.
Dopamine is critical for synaptic plasticity in the DMS; thus, it is crucial to consider estradiol's effect on dopamine as a mechanism for controlling LTP. Higher estradiol levels result in higher dopamine availability in the dorsal striatum, as evidenced by higher basal extracellular dopamine during estrus than during diestrus (Xiao and Becker, 1994) and by increased stimulated dopamine release in the striatum during estrus or after estradiol infusions (Becker and Beer, 1986;Becker, 1990;Castner et al., 1993;Becker and Rudick, 1999;Shams et al., 2016;Calipari et al., 2017;Song et al., 2019). This increased release may be an indirect result from estradiol's reduction of GABA release (Hu et al., 2006;Schultz et al., 2009). Despite the consensus on the effect of estradiol on dopamine release, the effect that enhanced dopamine has Research Article: New Research on plasticity is less clear. Heightened dopamine availability could have opposing effects on plasticity, depending on which downstream targets are affected. Corticostriatal LTP requires dopamine D1 receptor activity (Calabresi et al., 2000;Kerr and Wickens, 2001;Reynolds and Wickens, 2002;Hawes et al., 2013). On the other hand, D2 receptor activity induces long-term depression (LTD) through retrograde endocannabinoid signaling (Gerdeman et al., 2002;Kreitzer and Malenka, 2005). Perhaps the absence of LTP seen in estrus is because of increased dopamine binding to D2 receptors. Indeed, some studies have found that estradiol increases D2 receptor density (Fernández-Ruiz et al., 1989;Ursic et al., 2003); however, these effects are dependent on factors such as estradiol dosage and length of time applied (Roy et al., 1990;Bazzett and Becker, 1994). D2 receptors are involved in LTD through D2-receptor-containing SPNs (Calabresi et al., 1992;Kreitzer and Malenka, 2005) as well as through reduction of cholinergic interneuron activity, which can facilitate LTD in both D1R and D2R SPNs (Z. Wang et al., 2006). Future studies comparing LTP between identified D1-receptor-containing and D2-receptor-containing striatal neurons may be able to determine whether estradiol's effect on dopamine is responsible for the impairment in LTP.
There are a variety of other mechanisms through which ERa and GPER could be affecting LTP in the dorsal striatum. From research in males, corticostriatal LTP involves depolarization of SPNs, leading to calcium influx and subsequent activation of kinase pathways that cause LTP (Kreitzer and Malenka, 2008;Lovinger, 2010). Estradiol receptors could be targeting any of the components of this pathway. One possible mechanism is through reducing calcium influx through voltage-dependent calcium channels because of a reduction in excitability (Willett et al., 2020). This lower calcium would reduce the activation of calcium-dependent kinases critical for LTP, such as calcium/calmodulin-dependent protein kinase II (CaMKII; Q. Wang et al., 2017) and protein kinase C (PKC; Gubellini et al., 2004;Hawes et al., 2013). For more specific methods through which estradiol affects LTP, pathways activated by individual receptors, ERa and GPER, must be explored.
ERa coupling to mGluR may be its mechanism of action in inhibiting LTP. A subset of ERa activates mGluR5 (Grove-Strawser et al., 2010), which is a G q -coupled receptor. The level of calcium influx may determine whether Gq signaling leads to LTP or LTD (Kim et al., 2013). Under this model, a higher calcium level leads to processes that result in LTP via PKC activation, and a lower calcium level leads to processes that result in LTD via production of the endocannabinoid 2-arachidonoylglycerol (2-AG). As previously discussed, the lower excitability of SPNs during estrus could result in lower calcium influx in response to cortical input. Perhaps the ERa-mediated Gq activity in a lower-calcium environment results in the occurrence of LTD in some synapses after TBS, leading to no net increase in synaptic strength as measured by field recording. Another downstream target of mGluR5 is extracellular signal-regulated kinase (ERK; Choe and Wang, 2001;Yang et al., 2004;Mao et al., 2005), whose downstream targets lead to LTP (Xie et al., 2009;Cerovic et al., 2013;, as well as LTD (Cui et al., 2011) and depotentiation (Cerovic et al., 2015), although the conditions that determine the end result of ERK phosphorylation are unclear. This route indicates that ERa could be preventing bidirectional plasticity, and further investigation should be conducted into whether LTD occurs in the presence of ERa activation.
Another route through which LTP could be inhibited by ERa is through the ER's association with another type of mGluR. A separate subset of ERa activates the G i/ocoupled receptor mGluR3 (Grove-Strawser et al., 2010), which is located presynaptically on the terminals of cortical neurons and whose activation reduces calcium influx, and therefore glutamate release (Kupferschmidt and Lovinger, 2015). Less glutamate release would reduce the activation of postsynaptic glutamate receptors on SPNs involved in LTP induction, including AMPA and NMDA receptors, which are required for LTP (Calabresi et al., 1992;Partridge et al., 2000;Malinow and Malenka, 2002;Malenka, 2003;Lüscher and Malenka, 2012;Hawes et al., 2013). An ERa mediated increase in mGluR3 activity on cortical terminals could therefore be a separate mechanism through which ERa can affect LTP. ERa mediated changes in mGluR3 activity also may explain the reduced response to current injection seen in the input-output curve in the present study (Fig. 1B).
GPER's mechanism of action in inhibiting LTP could be either through its own G-protein activity or through the reinforcement of ERa activity. GPER is a Gs-coupled receptor (Thomas et al., 2010;Filardo and Thomas, 2012;Evans et al., 2016;Yu et al., 2018). While Gs activity is known to be involved in LTP through its downstream target protein kinase A (PKA; Spencer and Murphy, 2002;Hawes et al., 2013), a negative feedback loop involving elevated activity of phosphodiesterase (PDE) consequent to prolonged elevation of Gs activity could act paradoxically to reduce PKA activity during LTP (Lim et al., 1999;Houslay and Adams, 2003). Specifically, a higher basal level of Gs pathway activity throughout proestrus and estrus because of estradiol activation of GPER could result in enhanced PKA and PDE activity during estrus, which can reduce cAMP activity during TBS, thereby inhibiting PKA pathways required for LTP. Alternatively, GPER could be enhancing the activity of ERa. The interaction between these two receptors (Micevych and Christensen, 2012;Bourque et al., 2015;Acramel and Jacquot, 2022) could explain how either GPER activation or ERa activation inhibits LTP in diestrous females. ERa may be allosterically modulated by GPER to prevent LTP, which would explain why GPER inhibition alone is sufficient to rescue LTP. This mechanism may also explain why GPER activation did not prevent LTP in males; perhaps the estradiol level in males is too low to produce a high enough level of ERa activity for GPER modulation to have an effect. There are thus many possibilities through which these receptors could be inhibiting LTP, and this will be an important area of research moving forward, as it is crucial to understanding the complexity of estradiol's involvement in general synaptic dynamics underlying learning.
The LTP deficits uncovered by this study may underlie learning impairments in dorsal striatum-based behaviors observed in the presence of high estradiol. Dorsal striatal learning is underpinned by synaptic plasticity (Reynolds et al., 2001;Yin et al., 2009;Shan et al., 2014Shan et al., , 2015Hawes et al., 2015). The inhibition of LTP would be expected to result in poorer performance in learning tasks. Reflecting the current study's finding that ER activity inhibits LTP, past research has demonstrated that several types of learning that involve the dorsal striatum are impaired in the presence of higher estradiol levels. Rats given subcutaneous estradiol perform worse in cued learning (Galea et al., 2001;Daniel and Lee, 2004;V.C. Wang et al., 2008V.C. Wang et al., , 2009, response learning (Davis et al., 2005;Pleil et al., 2011), and spatial alternation (V.C. Wang et al., 2008Wang et al., , 2009Neese et al., 2010). All of these learning strategies are known to involve dorsal striatal circuits (Packard et al., 1989;Packard and McGaugh, 1996;Balleine, 2005;Yin et al., 2005a, b;Lee et al., 2008;Moussa et al., 2011). A particularly well-characterized example of estradiol's differential effects on learning is the switch in learning types from dorsal striatal-based learning strategies to hippocampal-based learning strategies. The bias in learning strategy between dorsal striatumbased learning and hippocampal-based learning fluctuates across the estrous cycle according to estradiol level, where higher estradiol stages favor place learning over response or cued learning (Korol et al., 2004;Yagi et al., 2017). This bias toward hippocampal strategies over dorsal striatal strategies can also be observed when estradiol is injected subcutaneously (Korol and Kolo, 2002;Davis et al., 2005;Quinlan et al., 2008;Hussain et al., 2016) or directly into the dorsal striatum (Quinlan et al., 2013). One study found that subcutaneous injection of the ERa agonist PPT alone is enough to produce this shift in learning strategy (Pisani et al., 2016). Estradiol also enhances other types of hippocampal-based learning such as object placement and recognition memory (Walf et al., 2006;Paris and Frye, 2008;Inagaki et al., 2010Inagaki et al., , 2012Phan et al., 2012Phan et al., , 2015Jacome et al., 2016;Luine et al., 2018). Consistent with this switch, estradiol is necessary for and enhances synaptic plasticity in hippocampus (S.G. Warren et al., 1995;Foy et al., 1999;Smith and McMahon, 2006;Grassi et al., 2011;Hasegawa et al., 2015;Lu et al., 2019). In addition, excitability and spine density in the hippocampus both fluctuate across the estrous cycle, with highest excitability during proestrus and estrus, and higher spine densities observed during stages with high estradiol and progesterone (Kubo et al., 1975;Woolley and McEwen, 1992;Scharfman et al., 2003;Kato et al., 2013;Jaric et al., 2019). Therefore, the results of the current study are congruous with the growing body of literature indicating that estradiol's effect on synaptic plasticity parallels its effect on the learning types that involve this plasticity. In other words, estradiol may be a regulator of behavioral strategy through control of synaptic plasticity, e.g., selecting between hippocampal and striatal strategies depending on high versus low estradiol, respectively.
The relevance of these results to behavior may extend to pathology as well. A major component of striatal-based behavior that is influenced by estradiol is habit formation, which contributes to addictive behaviors. Among individuals in treatment for drug abuse, women are observed to develop habitual drug use more quickly than men (Piazza et al., 1989;Becker et al., 2017; and can exhibit different levels of craving and relapse across the menstrual cycle (Allen et al., 2009;Franklin et al., 2015;Carlson et al., 2017;Hayaki et al., 2020;J.G. Warren et al., 2021). In rodent studies, chronic estradiol treatment leads to quicker acquisition of cocaine, nicotine, and methamphetamine self-administration than in estradiol-depleted females (Jackson et al., 2006;Larson et al., 2007;Kucerova et al., 2009;Flores et al., 2016). These effects, however, are dose and drug dependent, as increasing estradiol doses can reduce cocaine intake , and estradiol-depleted rats exhibit higher opiate self-administration than those treated with estradiol (Sharp et al., 2021). During habit formation, behavior changes from goal-directed to goal-independent (habitual) as control over drug intake switches from the DMS to the dorsolateral striatum (DLS; Corbit et al., 2012;Malvaez, 2020). It has been hypothesized that the control does not simply shift, but that there is a competition between these two subregions, where stronger connections in one subregion will bias dominance over behavior to that subregion. Indeed, inhibiting the DLS produces a return to goal-directed behavior and inhibiting the DMS produces habitual behavior (Yin and Knowlton, 2006). Perhaps higher estradiol promotes the development of habitual behavior as it weakens DMS-based, goal-directed learning. The effect of estradiol in the DLS of females is unknown, but in males it has been demonstrated to be necessary for LTP (Tozzi et al., 2015). Estradiol's action on the mechanisms underlying learning in the dorsal striatum may be a key component in understanding the hormone's role in substance use disorders.